It is widely known that biological functions of a variety of proteins are each expressed when a peptide chain having a primary sequence of amino acids intrinsic to each protein forms a unique three-dimensional structure according to the primary sequence of the amino acids. Hence, information on three-dimensional structures of proteins are quite important intellectual properties, and collected in an international information collecting institution (Protein Data Bank (hereinafter may also be referred to as PDB); for example, see, “RCSB Protein data bank”, [online], the Research Collaboratory for Structural Bioinformatics, [retrieved on Heisei 24.7.25 (Jul. 25, 2012)], Internet (http://www.pdb.org/pdb/home/home.do)) and so forth. It is widely used in medicine, biology, and other basic researches, and in drug development and other applied researches.
Currently, the most potent method as an experimental approach for determining the three-dimensional structure of a protein is X-ray crystallography that utilizes an X-ray diffraction of a single crystal of a protein. Not less than 90% of information on three-dimensional structures of proteins collected every year in PDB are obtained from this X-ray crystallography.
On the other hand, an NMR technique which is a novel protein structure determination approach having been introduced in 1980s accounts for only 10% or less of information on three-dimensional structures provided to PDB and so forth until now. Nevertheless, this method is capable of determining the structure of a protein not in a crystallized state but in such a state that amino acid residues in the protein can freely move around in an environment similar to an environment where the protein plays its biological function, such as in an aqueous solution, micelle, and lipid bilayer. Since the method can provide so-called “dynamic structural information,” there is information provided entirely different from “static structural information” generally obtained by the X-ray crystallography.
As described above, to be able to utilize dynamic structural information in addition to static structural information is a great advantage in making use of information on the three-dimensional structure of a protein in basic researches and applied researches. Hence, the development of novel NMR analysis techniques has attracted great interest worldwide, and various technical innovation competitions are taking place all over the world.
In this respect, the X-ray crystallography has already had satisfactory results as a structural analysis method for almost half a century and has been technically matured. On the other hand, only 20 years have passed since the NMR technique was introduced as a structural analysis method and the NMR technique still has a lot of problems to be solved. Among such problems, a problem sought to be solved as soon as possible is that proteins successfully subjected to structural analysis according to the NMR technique are limited to proteins having relatively low molecular weights.
In fact, the molecular weight of proteins whose structures have been specified by the NMR technique and registered in PDB is roughly 10,000 to 20,000. To solve such a problem in the present situation, technical improvements have been made in each of three basic technical fields such as (1) preparation technique of a protein sample labeled with a stable isotope, (2) multi-dimensional, multi-nuclear NMR measurement technique, and (3) three-dimensional structural analysis technique utilizing NMR spectrum information. Recently, a three-dimensional structure of a protein having a molecular weight of approximately 20,000 to 25,000 has also been successfully determined by utilizing NMR techniques. However, all of the NMR techniques utilizing the improved techniques so far are based on the premise that the structural analysis precision is sacrificed to some extent in order to determine the structure of a protein having a higher molecular weight. For this reason, regarding information on the three-dimensional structure of a high-molecular-weight protein obtained by an NMR technique utilizing conventional techniques, the precision of the structural analysis is insufficient, and such a precision problem in the structural analysis has been a major obstacle when obtained information on a three-dimensional structure is utilized in the fields of drug development and the like.
The present inventors have already demonstrated that developing a method for optimizing a technique associated with all of the above basic technical fields (1) to (3) makes it possible to develop a technique enabling a structural analysis at a level far higher than the molecular-weight limitation in the NMR technique, and also to achieve a great improvement in the precision of a structural analysis and a great reduction in the analysis time (see International Publication No. WO2003/053910). This novel technique later designated as the SAIL (Stereo-array isotope labeling) method by the present inventors has made it possible, as an innovative new technique, to increase the range of the molecular weight that can be successfully subjected to NMR structure analysis to a range of approximately 40,000 to 50,000, and simultaneously to increase the precision of the structural analysis. The basic idea of the SAIL method is to greatly reduce the number of hydrogen atoms (1H) in amino acid residues constituting a protein without reducing the amount of information on a three-dimensional structure to be obtained, so that the structural information is obtained quickly with higher precision. Recently, a Canadian NMR research group has reported that, among amino acid residues forming a hydrophobic core of a protein, selectively forming only methyl groups of leucine (Leu), valine (Val), and isoleucine (Ile) as 13C1H3 and deuterating all the remaining hydrogen atoms make it possible to observe sharp NMR signals of the methyl groups of these amino acid residues even in a high-molecular-weight protein having a molecular weight exceeding 100 kDa, and to determine a fold structure of a peptide chain of the protein. Although the precision of obtained structural information is insufficient, this approach makes it possible to prepare a sample easily by utilizing readily available methyl-labeled amino acids described above, and to apply an NMR technique to proteins having high molecular weights, which has been thought to be impossible heretofore. Accordingly, the application of this approach is increasing as an innovative technique.
On the other hand, in International Publication No. WO2007/099934, the present inventors have developed stable isotope-labeled amino acids for more reliable and higher sensitivity assignment of 13C and 1H signals of amino acid residue side chains. In these stable isotope-labeled amino acids, a methylene proton (1H) on a side chain is isolated by deuterating a neighboring hydrogen atom, thereby enabling high sensitivity observation of an NMR signal derived from the methylene proton. Simultaneously, signal assignment can be easily achieved by spin couplings of 3J (13C—13C), 3J (13C—1H), or the like.